A combined in situ hybridization and immunofluorescence procedure allowing visualisation of peptide mRNA and serotonin in single sections.

We describe a novel procedure for combining immunocytochemistry with in situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridization and is particularly suitable for antigens which are labile or sensitive to in situ hybridization processing. We have evaluated the technique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing 35S-labelled oligonucleotide probes. Successful double labelling was obtained and showed that galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive cells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic cell population. Double-labelled preparations produced using this technique can be conveniently viewed using epipolarised combined with epifluorescent illumination. Careful analysis of procedural variables revealed that it is not possible to carry out high-sensitivity 5-HT immunocytochemistry following in situ hybridization. The immunostaining is much poorer on slide-mounted sections than on free-floating sections, and 5-HT appears to be lost during the in situ hybridization steps of dehydration/delipidation and incubation in hybridization buffer. The procedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.